While eradicated as an endemic disease, smallpox remains a threat to human health because it may be used by bioterrorists. The potential spread of variola virus or recombinant forms of variola or other poxviruses requires the development of novel therapeutic approaches. We propose to develop and utilize RNA interference (RNAi) technology to treat smallpox. RNAi is accomplished by the introduction of double-stranded RNA into the cell, resulting in sequence specific degradation of the target mRNA. Application of short (21-25 bp) double stranded RNA (siRNA) also induces effective RNAi and limits non-specific effects. We have recently demonstrated that siRNA can be efficiently introduced into mammalian cells in vivo, and can be used to modulate gene expression. In this SBIR Phase I grant proposal, we will determine if poxvirus gene expression can be inhibited by siRNA. For these experiments, we will use vaccinia virus as a highly relevant model for variola virus. Several genes from different viral pathways (transcription, replication, virion formation, virulence) will be targeted in vitro. Effects upon target gene expression and viral replication will be measured. These experiments should demonstrate the feasibility of using siRNA for smallpox therapy or prophylaxis.